Automated fingerprint methods and chemistry for product authentication and monitoring

ABSTRACT

Highly efficient, low cost, methods and compounds to determine product authenticity, tampering, manufacturing compliance are provided. The methods and chemicals defined are capable of measuring the relative amount of key materials in these products. The compounds are light emitting and interact with key elements in products like, neutral spirits, vodka, tequila, soft drinks and infant formulas. After the interactions are complete, methods are employed to determine resulting key components by light emission.

BACKGROUND OF THE INVENTION

This application is a division of Ser. No. 09/191,947 filed Nov. 13,1998, now U.S. Pat. No. 6,232,124, which is a continuation-in-part ofSer. No. 08/969,194 filed Nov. 13, 1997, now abandoned, which is acontinuation-in-part of Ser. No. 08/852,108 filed May 06, 1997, nowabandoned, which is a continuation-in-part of Ser. No. 08/642,927 filedMay 06, 1996, now U.S. Pat. No. 5,753,511.

This invention is in the general field of methods, reagents, andapparatus for authenticating or monitoring sample composition.

Authenticating and monitoring products to discriminate between verysimilar complex mixtures is useful for various reasons. First, the useof counterfeit substances (e.g., misbranded material from a competitoror misformulated material from a licensee/franchisee) should be detectedto preserve the integrity of a brand.

Characteristics of a product can be used to identify its lot. Similarmethods can be used in quality control tests. Also, productcounterfeiting raises serious health and safety issues. In 1995, acounterfeit-labeled version of infant formula reportedly was distributedto 15 states in the continental United States. Counterfeit wine,spirits, perfume, infant formula, soft drinks, cosmetics, andpharmaceuticals are estimated to cost United States businesses 200billion dollars per year (“The Boston Phoenix,” Section One, Dec. 2,1994).

It is important to develop rapid, cost effective, and enforceablemethods to identify fraudulent or tampered products. It is alsoimportant to determine manufacturing compliance using automated methodsto decrease the amount of time spent identifying fraudulent products. Itis desirable to minimize the time required from highly skilledresearchers and technicians to conduct and record the results ofon-line, off-line, and off-the-shelf product authenticity/compliancetests.

There have been attempts to determine product (e.g., infant formula)authenticity by protein electrophoresis, which requires substantial time(and expense) for set up and analysis. In other industries, e.g. wineand spirits, Fourier-transform infrared analysis, gas chromatography,pH, Raman spectroscopy and other analytical methods have been used orproposed for product authentication (Constant et al., Differentiation ofAlcoholic Beverages FT-IR Spectra. An Original Multivariate Approach,ACS Abstract presented at 208th ACS National Meeting, Aug. 25, 1994,published in the Issue of Chemical and Engineering News, 10 1994).

Biocode, Limited has used fluorescent labeled antibodies to determineingredients in products.

U.S. Pat. No. 5,429,952 discloses adding light-emissive chemicals to aproduct for analysis, as exogenous product tags which do not ordinarilyform part of the product.

The use of standard analytical methods to monitor every lot or batch fora product or competitor product for authenticity or compliance withlaboratory equipment can often be costly.

SUMMARY OF THE INVENTION

I have discovered an automated method of developing a database to storeinformation for “fingerprint”-type analysis of products (even as toproduct lot numbers and batch). The automated analysis is a method ofevaluating and discriminating products, even within a narrow field orindustry, competing and otherwise, e.g., to establish authenticity orpoint of origin of the product. The invention relates to a method foridentifying analytes such as key ingredients and/or the relative amountsof analytes such as key ingredients in products. The method allows forauthenticating and monitoring products for fraud and quality controlusing light emission. The invention also relates tolight-emissive-compounds (e.g., including one or more light emissivecompounds) which can be used to identify and quantitate the relativeamounts of analytes in products.

In general, the methods pertain to obtaining an emission profile of asample. An “emission profile”, as used herein, refers to data collectedrelating to emission, for example, emission intensity or time ofemission. The data collected for the emission profile can be expressedin relative terms, e.g. relative emission intensity or time of relativeemission, when data for the sample and a standard and/or a control arecompared.

In one aspect, the invention features a method for determiningrelatedness of a sample to a standard known to be authentic or known tohave at least one selected characteristic of authentic material. Themethod includes: a) providing a mixture of sample and least onelight-emissive compound (“LEC”); (b) irradiating the sample mixture withan irradiating wavelength of light; (c) monitoring at least one emittedwavelength of light (generated in response to the irradiating) toestablish a sample emission profile; and (d) providing a standardfingerprint characteristic of a standard mixture; and (e) comparing thesample emission profile with the standard fingerprint to determinewhether the sample is authentic. The standard mixture includes thestandard and the light-emissive compound. The standard fingerprint isgenerated by irradiating several of the standard mixture with theirradiating wavelength and monitoring the emitted wavelength in responsethereto.

In preferred embodiments, two and preferably three or morelight-emissive compounds are employed, and a fingerprint profile ofseveral light-emissive compounds is compared to the correspondingemission intensities for the sample. Most preferably, the light-emissivecompounds emit light at nonoverlapping wave lengths, whereby multiplecompounds can be added to the sample and/or standard at the same time.

In preferred embodiments, the method further includes: providing abackground control mixture which includes the light-emissive compoundwithout the sample or the standard; irradiating the background controlmixture with the irradiating wavelength and monitoring the emittedwavelength in response thereto, to establish background emission; anddetermining the emission profile of the sample based on at least onedifference between the emission of the control mixture and the emissionof the sample mixture. It is preferred that the standard be acomposition having a predetermined relative amount of a componentcharacteristic of authentic material. The sample fingerprint isgenerated based on a first change in emission, determined by comparingthe background emission and the emission from the sample mixture. Thestandard fingerprint is generated based on a second change in emissions,determined by comparing the background emission and the standardemission for each measurement. The comparing step includes comparing thefirst change in emission to the background adjusted fingerprint, e.g.,to quantify relative amounts of sample component.

In another aspect of the invention, a method is provided for determiningwhether a product is authentic. A liquid sample of a test product isobtained and a light emissive compound then is added to the liquidsample to form a test sample. The light emissive compound interacts withan analyte of the product. The test sample is irradiated, and theintensity of light emitted from the test sample at a wavelength isdetermined. The intensity of light emitted from the test sample at thiswavelength then is compared to the intensity of light emitted at thewavelength as a result of irradiating a mixture of the light emittingcompound and an authentic liquid standard of the product, whereinsimilarity of light emission intensity is determinative of authenticityof the sample and this similarity of light emission intensity isdeterminative of nonauthenticity of the sample. In one importantembodiment, the intensity of light emitted from the test sample iscompared to the intensity of light emitted from a plurality of themixture, and wherein authenticity requires the intensity of lightemitted from the test sample to be within a pre-selected confidencelimit defining a range of intensity calculated from the intensity oflight emitted from the plurality of said mixture. The plurality of saidmixture is at least four standards containing a mixture of the lightemitting compound and an authentic liquid standard of the product, andpreferably is four such mixtures.

In certain of the foregoing embodiments, the chemical composition of theproduct is unknown. In other of the embodiments, the chemical structureof the analyte to which the light emitting compound binds is unknown. Instill other embodiments, the analyte is other than an exogenous producttag. In one particularly important embodiment, the product is a liquidconsumable product.

As mentioned above, a plurality of light emissive compounds can be used.In such embodiments, it is preferred that each light emitting compoundbinds to a different analyte of the product. Most preferably, the lightemissive compounds is a fluorescent dye.

In other preferred embodiments, the light-emissive compound is added tothe sample by an automated pipette. It is preferred that the samplemixture be dispensed by an automated pipette in a multiwell plate.

In other preferred embodiments, the standard, the sample, or both,inherently include a fluorescent, phosphorescent, or luminescentcompound. In some products the compound is caffeine.

In other preferred embodiments, the light-emissive compound isfluorescent, phosphorescent, or luminescent, and emission varies inresponse to quantity or quality of product analytes. Preferably, thelight-emissive compound interacts with components of the sample, thestandard, or both, to yield at least one fluorescent, phosphorescent, orluminescent component.

In other preferred embodiments, the standard is a composition having apredetermined relative amount of an analyte characteristic of authenticmaterial, and the comparing step includes quantifying the relativeamounts of the analyte in the sample.

In preferred embodiments, the method includes performing steps (b)-(c)described above, at least two times and preferably three times. Steps(b)-(c) may be performed using the same or different light-emissivecompounds, and the same or different irradiating and emissionwavelengths are monitored in each performed step.

In one important embodiment, the standard is a caffeine-containingbeverage, and the light-emissive compound is: a)5-(2-carbohydrazinomethylthioacetyl)aminofluorescein; b)5-(4,6-dichlorotriazinyl)aminofluorescein; c)Fluo-3 pentaammonium salt(Minta et al., J. Biol. Chem. 264:8171, 1989 and U.S. Pat. No.5,049,673); d) 4-aminofluorescein; e) 5-aminofluorescein; f) sulfiteblue coumarin; g) courmarin diacid cryptand (CD222) (Costlei et al., J.of Chem. Society Perkins translation 2, p. 1615); or h) Eosin Y.

In another important embodiment, the standard is an infant formula, andthe light-emissive compound is selected from the group consisting of5-(2-carbohydrazinomethylthioacetyl) aminofluorescein,5-(4,6-dichlorotriazinyl)aminofluorescein, Fluuo-3 pentaammonium salt,or Courmarin benzothiazole, tetrapotassium salt (BTC5N) (Cell Calcium,p. 190, 1994). In other preferred embodiments, the standard containscorn syrup, and the light-emissive compound is selected from the groupconsisting of 5-(2-carbohydrazinomethylthioacetyl) aminofluorescein,5-(4,6-dichlorotriazinyl)aminofluorescein, Fluo-3 pentaammonium salt,4-aminofluorescein, 5-aminofluorescein, sulfite blue coumarin, courmarindiacid cryptand (CD222), or Eosin Y. In other preferred embodiments, thestandard is an ethanol-containing beverage and the light-emissivecompound is selected from the group consisting of5-(2-carbohydrazinomethylthioacetyl)aminofluorescein,5-(4,6-dichlorotriazinyl)aminofluorescein, Fluo-3 pentaammonium salt,proflavine hemisulfate, tetra(tetramethylammonium) salt, acridine orangehydrochloride hydrate, BTC-5N, acriflavine, 4-aminofluorescein, or5-aminofluorescein. Compound 11 is sulfite blue coumarin compound 12 iscourmarin diacid cryptand (CD222). Compound 13 is Eosin Y. In otherpreferred embodiments, the standard is an aqueous mixture, and thelight-emissive compound is a compound that interacts or reacts withheavy metals, the light-emissive compound being selected from the groupconsisting of Fluo-3 pentaammonium salt, or BTC-5N.

In another aspect, the invention features a method for determiningrelatedness of a first sample to a second sample, neither of which is aknown standard. The method includes: (a) providing a first samplemixture including the first sample and at least one light-emissivecompound; (b) irradiating a plurality of the first sample mixture withan irradiating wavelength of light; (c) monitoring at least one emittedwavelength of light generated in response to the irradiating, toestablish a first sample fingerprint characteristic of the first samplemixture; (d) providing a second sample fingerprint characteristic of asecond sample mixture, the second sample mixture including the secondsample and the light-emissive compound; the second sample fingerprintbeing generated by irradiating a plurality of the second sample mixturewith the irradiating wavelength and monitoring the emitted wavelength inresponse thereto; and (e) comparing the first sample fingerprint withthe second sample fingerprint to determine relatedness of the twosamples.

In preferred embodiments, the first sample is identified as a specificproduct or as part of a homogeneous lot of a product by comparing thefingerprint profile or emission profile of the first sample to a libraryof fingerprints of samples whose product composition or lot number areknown.

In other preferred embodiments, the method further includes providingone or more additional fingerprints to generate a fingerprint profilefor each of at least two additional light emissive compounds andcomparing the first sample mixture fingerprint profile to the secondsample or standard fingerprint profile.

In preferred embodiments, the method is used to determine productauthenticity, product tampering or product manufacturing compliance. Inother preferred embodiments, the sample is a perfume, fragrance, flavor,food, or beverage product.

In another aspect of the invention, a method is provided for selecting adye for determining authenticity of a product. A candidate dye is addedto a plurality of candidate dilutions of a liquid sample of an authenticstandard of the product, the candidate dye being light emissive at aparticular wavelength when irradiated if it interacts with an analyte inthe liquid sample. A test dilution then is selected at which thecandidate dye emits light at a selected intensity when said candidatedye is added to said liquid sample at the test dilution. A range ofintensity of light emission at discrete wavelengths is determined for aplurality of mixtures of said candidate dye and said liquid sample atsaid test dilution. An experimental intensity of light emission at thediscrete wavelengths is then determined for a mixture of said candidatedye and a liquid sample of nonauthentic product at said test dilution.Finally, the experimental intensity at the discrete wavelengths iscompared to the range of intensity of light emission at the discretewavelengths, said dye being selected as useful for determiningauthenticity of said product if said experimental intensity fallsoutside of said range of light emission at the discrete wavelengths. Inone embodiment, the candidate dye is a plurality of candidate dyes, eachof the dyes emitting light at different wavelengths, and wherein theanalyte is a plurality of analytes, each dye binding to a different ofsaid plurality of the plurality of analytes. In an important embodiment,the chemical composition of the product is unknown and/or the chemicalstructure of the analyte is unknown. In other important embodiments, theproduct is a liquid consumable product.

In another aspect of the invention, a computer implemented method fordetermining authenticity of a liquid product is provided. The methodinvolves receiving light emission data produced by adding a component toa test sample of the liquid product and measuring light emissiontherefrom. It also involves receiving light emission data produced bymeasuring light emission from a sample of a mixture of an authenticliquid product and the component. There then is a comparison of theintensity of light emission from the test sample to intensity of lightemission from samples of the plurality of the mixtures, whereinauthenticity requires the intensity of light emission from the testsample to be within a preselected confidence limit defining a range ofintensity at discrete wavelengths calculated from the intensity of lightemission at the discrete wavelengths from the plurality of the mixtures.

In one important embodiment, a computer database is used for storing andmaking available information about light emission of an authenticproduct. The database includes a computer-readable medium having acomputer-readable logic stored thereon, wherein the computer-readablelogic comprises a plurality of records for the authentic productindicating measurements of intensity of light emitted by samples of aplurality of mixtures of the authentic product with a component. Thedatabase also includes an indication of the component, wherein therecords are accessible using an indication of the component and/or theauthentic product wherein the step of receiving light emission data forthe authentic product includes the step of accessing thecomputer-readable medium using an indication of the component and/or theproduct to retrieve the records.

In another aspect of the invention, a computer database for storing andmaking available information about light emission of an authenticproduct is provided. The database included a computer-readable mediumhaving computer-readable logics stored thereon, wherein thecomputer-readable logic comprises a plurality of records for theauthentic product indicating measurements of intensity of light emittedby samples of a plurality of mixtures of the authentic product with acomponent, and an indication of the component. Also included are meansfor accessing the computer-readable medium using an indication of thecomponent and/or the authentic product to retrieve the records.

It is a feature also of the present invention that, when adding alight-emitting compound to a sample in accordance with the methodsdescribed herein, the sample can be separate from the standard. Thisdiffers from the situation where product tags are used, in that producttags are added to an authentic product to form a tagged mixture whereinthe addition of the tag to the sample is not separate from the additionof the tag to the standard.

Light-emissive compounds are involved in light emission in response toirradiation with light of a different wavelength. Light emission ofinterest can be a result of phosphorescence, chemiluminescence, or, morepreferably, fluorescence or polarized fluorescence. Specifically, theterm “light emissive compounds,” as used herein, means compounds thathave one or more of the following properties: 1) they are a fluorescent,phosphorescent, or luminescent; 2) interact with components of thesample or the standard or both to yield at least one fluorescent,phosphorescent, or luminescent compound; or 3) interact with at leastone fluorescent, phosphorescent, or luminescent compound in the sample,the standard, or both to alter emission at the emission wavelength. Theemission wavelength can be any detectable wavelength including visible,infrared (including near infrared), and ultraviolet. Light, as usedherein, likewise can be of any wavelength.

Light-emissive compounds also include compounds that cause, or interactwith components of the standard or sample to cause, or alter, RamanScatter at a scatter or emission wavelength. The Raman effect occurswhen light from a strong source (typically a laser) interacts with amaterial. Most of the light is absorbed or scattered without wavelengthchange but some of the light is scattered into other wavelengths (theRaman scatter).

“Fingerprint” refers to the data set of light emission intensity from alight-emissive compound in combination with a liquid sample of a productmeasured; at least three times, three such combinations measured atleast once, or both. Accordingly, each product can have a particularfingerprint. A “fingerprint profile” is an assembly of fingerprints of aliquid sample of a product in combination with a series (or profile) ofdifferent light-emissive compounds.

As noted above, the emission profile can include, but is not limited to,emission intensity and time of relative emission. The same informationthat can be derived about the amount and/or concentration of analytes byemission intensity measurement also can be derived from measurement ofthe time of emission, e.g. the time of relative emission of fluorescentcompounds in a sample. Analysis of the emission intensity or time ofrelative emission can be done as described herein and by other methodsknown to one of ordinary skill in the art. Other emission propertiesmeasurable by one of ordinary skill in the art (e.g. emission half-life,emission decay characteristics) are also embraced in the term emissionprofile.

The term “analyte”, as used herein, means a key ingredient or tracecompound of the product. A native analyte is one which is ordinarilyfound in the unadulterated product, not added as an exogenous producttag. The invention relies upon interaction of light emissive compoundswith such analytes, whereby alterations in a product can be detected,including (1) dilution of an analyte, (2) substitution of an ingredientfor an analyte, (3) addition of a compound which alters interaction ofthe light emissive compound with an analyte and (4) addition of acompound which quenches light emission resulting from interaction of alight emissive compound with an analyte. Most frequently the alterationdetected is in the amount of analyte bound to the light emissivecompound, which is reflected by the intensity of light emitted when asample is irradiated.

By “interacts with”, as used herein, it is meant reacting,intercalating, binding or any other interaction which causes the dye toalter its light emission properties when irradiated.

The term “key ingredient,” as used herein, means a component included ina composition of a product that is important in identifying theparticular product.

The term “trace compound,” as used herein, means a compound that ispresent in low concentrations (e.g., at ppm or ppb levels) in a product.The trace compound can be related, for example, to a particular keyingredient. The trace compound can be introduced at the source of thekey ingredient or during the manufacture of the product.

The invention can include one or more of the following advantages. Themethod can be used in the distilled spirits industry, where tracecompounds and key ingredients can be measured using specificlight-emissive compounds. Further, light-emissive compounds thatindicate the source of ethanol can be used to determine the authenticityof a product. For example, spirits derived from yellow dent corn containdifferent trace compounds than spirits derived from cane sugar.

Moreover, although colas, and other soft drinks, contain similar levelsof key ingredients, the levels key ingredients can be used to determinewhether a particular manufacturer is diluting the concentrate to theappropriate level. For example, caffeine can be a targeted ingredientfor light-emissive compounds in the analysis of soft drinks. Additionaltargets in soft drinks can include, but are not limited to, the highfructose corn syrup and the pH.

Furthermore, perfumes, fragrances, flavors, foods, and all types ofbeverages can be fingerprinted, using the methods of the invention,without adding any reagents to the product the user is going to consume.An advantage of invention is that exogenous product tags need not beadded. Instead, native analytes of the product can be assayed. This isparticularly important in determining authenticity of food products,where it is undesirable to add tags which could affect taste, odor,consistency and the like and might even be harmful to health wheningested. This is of great importance to many companies which arereluctant to adulterate their products.

The invention also is useful in identifying pharmaceutical activeingredients and/or excipients. The invention, therefore, can be used toauthenticate pharmaceutical or other chemical products. In the instancewhere a fingerprint is obtained for a pharmaceutical formulation, anidentical fingerprint may permit an inference that the pharmaceuticalformulation was prepared by a particular process, which itself may be apatented process. In addition, there may be unique ingredients used in apatented process, the presence and concentration of which can be used todetermine the authenticity of a material manufactured by that process(when the material contains trace levels of the unique ingredients) oras evidence of infringement of the patented process. Thus, single ormultiple dyes can be selected or developed to identify compounds orexcipients that would be present (or present at particularconcentrations) only as a result of performing a patented process.

The invention allows accurate light-emissive profiles of products to bedetermined and monitored without altering the product.

Another advantage of the invention is that it is unnecessary to know ordetermine the composition of the product in order to select lightemissive compounds and to develop assays for determining accuratelyauthenticity. Thus, it is unnecessary to know or determine the formulafor Coca-Cola® or Pepsi® in order to test the authenticity of productssold under those trademarks. This is to be contrasted with many infraredmethods (e.g., near IR, mid IR and Fourier Transform IR), that oftenresult in gathering sufficient information to determine the compositionof a product being tested. This advantage of the present invention is ofgreat importance to companies reluctant to identify the secretingredients of their products.

A further advantage of the invention is that the use of light-emittingcompounds results in a sensitivity level that far exceeds thesensitivity levels achievable by the use of Fourier Transform IRmethods.

Other features and advantages of the invention will be apparent from thefollowing detailed description thereof, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing that shows the chemical structures of Compounds1-13.

FIG. 2 is a data flow diagram.

DETAILED DESCRIPTION OF THE INVENTION

The invention features an automated method for analyzing analytes suchas key ingredients and the relative amounts of analytes such as keyingredients in products which in turn enables authentication andmonitoring products for fraud and quality control. Particularlight-emissive compounds can be used to identify and quantitate therelative levels of analytes such as key ingredients in the products.

One method for identifying counterfeit or altered products relies on thedevelopment of a group of between two and seven specific light-emissivecompounds for a single product along with specialized automated handlingmethods and new data analysis. These methods can be used to provide amethod which is simpler to use than prior techniques and which can beperformed rapidly using conventional and generally available equipment.It is a further aspect of the invention to provide a technique whichgives quantitative measure of the degree to which the product is alteredor tampered with. It is a further aspect of the invention to providemethods and compounds for identifying key-product ingredients.

The invention provides a method for determining the relative amounts ofanalytes in a product by exposing the products to selectedlight-emissive compounds present in a light-emissive compound. Analytesare selected so that in the presence of the analyte, the compounds caninteract with (e.g. partition, intercalate, or bind to) the analytes inthe aqueous and/or organic liquid fractions of the product. Theinteraction between the components of the product and the light-emissivecompound induces a chemical change that can be detected using automatedlight emissive detection systems. Light-emission can includeluminescence, fluorescence, or phosphorescence. Fluorescence isdescribed, for example, in “Practical Fluorescence,” Second Edition, G.G. Guilbault, Editor, Marcel Dekker, Inc., 1990, which is incorporatedherein by reference.

In general, a sample of the product and the light emissive compound aremixed. The light-emissive compounds and analytes in the product areallowed to react for a period of time and temperature that is specificfor each product and light-emissive compound, for example, until lightemission from the mixture no longer changes with time. Bandpass andcutoff filters are used to isolate excitation wavelengths from emissionspectra due to light emission from the sample. Change in light emissiondue to the interaction can be determined, from the formula[(Fd−Fp)/Fd]×100, where the light emission of the light emissivecompound in the absence of product is Fp, and the light emission afterexposing the light-emissive compound to the product is Fd. The lightemission changes as a result of interactions of the light-emissivecompound with analytes in the product. Light emission also can changedue to indirect influences, such as quenching by an ingredient of anadulterated product.

The light-emissive compound can include two light-emissive compounds andcan be added together in the same sample well, if the emission maximumof the dyes is more than 40 nm apart. The wavelength filter must bechanged for each light-emissive compound being observed. There is nopractical limit to the number of light-emissive compounds that can beused to demonstrate the specific presence of a particular analyte. Thenumber of light-emissive compounds can be increased to indicate thespecific presence of an ingredient or to rule out possible non-specificanalysis of closely related compounds.

It is possible to determine the authenticity of product if the trace orchemical structure of the analyte or product is unknown, preferably byusing between three and seven individual light-emissive compounds in thelight-emissive compound. Using an automated robotics workstation (e.g.,the Beckman Biomek 1000), it is possible to combine the light-emissivecompounds in random order with the product standards in a microwellplate. Once a detectable light emission pattern is developed for all thestandards, a single test product can be added to the same microwellplate (e.g., up to 99 standards and 1 single test product). The lightemission output of the sample is compared to each of the standards onthe plate run. In this way, it is possible to determine authenticitywithout developing a prior record of standard light emission levels.

There are many examples of light-emissive compounds that can be includedin the light-emissive compound, some of which are shown in FIG. 1(Compounds 1-13). Compound 1 is5-(2-carbohydrazinomethylthioacetyl)aminofluorescein. Compound 2 is5-(4,6-dichlorotriazinyl)aminofluorescein. Compound 3 is Fluo-3pentaammonium salt. Compound 4 is proflavine hemisulfate(3,6-dimetliylaminoacridine hemisulfate). Compound 5 istetra(tetramethylammonium) salt. Compound 6 is acridine orangehydrochloride hydrate. Compound 7 is BTC-SN. Compound 8 is acriflavine.Compound 9 is 4-aminofluorescein. Compound 10 is 5-aminofluorescein.Compound 11 is sulfite blue coumarin. Compound 12 is courmarin diacidcryptand (CD222). Compound 13 is Eosin Y.

Infrared light emissive compounds, including those emitting in the nearinfrared wavelengths, can improve performance in certain circumstancesbecause of the low endogenous background fluorescence of many materialsin the infrared region. The addition of infrared dyes allows for asignificant increase in certain circumstances in signal to noise ratioin an aqueous environment. Examples include porphyrins, cyanines,naphthoquinone methides, squarylium dyes, polymethines, etc. Most commonare the cyanines including metal-containing phthalocyanines,pentamethines, naphthalocyanines, merocyanines, tricarboncyanines,indocyanines and isothiocyanato-functionalized cyanines.

Some examples of the liquid products that can be analyzed and thelight-emissive compounds that can provide distinctive and significantanalyses of the products are: alcohol-based products such as neutralspirits, vodka, and tequila that can be analyzed, for example, withCompounds 1, 2, 3, 4, 5, 6, 7, 8, or 9; sucrose and high fructose basedproducts such as soft drinks (e.g., Coca-Cola and Pepsi) that can beanalyzed, for example, with Compounds 1, 2, 3, or 9; and infant formulassuch as Similac, Carnation, Enfamil that can be analyzed, for example,with Compounds 1, 2, 3, or 7. It should be understood that a liquidsample may be obtained from a liquid product or from nonliquid products(e.g., by dissolving a solid or semisolid, by extraction of a solid anddissolution of the extracted material or the like).

The methods of the invention can be used to analyze other liquidproducts as well as liquid samples derived from other products, based onthe correct choice of light emissive compounds used in the analysis. Forexample, light-emissive compounds that are amine-containing (e.g.,Compound 1) and light-emissive compounds that are reagents for modifyingamines, alcohols, arginine, guanosine,' and polysaccharides (e.g.,Compound 2) can be used in product authenticity/monitoring and testingof, for example, neutral spirits, distilled spirits, infant formula, orsoft drinks. In addition, light-emissive chemicals that are calciumindicators (e.g., Compound 3) or are capable of complexing with Cd²⁺, Zn²⁺, Pb ²⁺ and Ba ²⁺ (e.g., Compound 7) can be used for productauthenticity/monitoring testing of neutral spirits, distilled spirits,or soft drinks. Light-emissive acridine compounds (e.g., Compound 6) arecapable of complexing with lipids and fats for product authentication ormonitoring of distilled spirits or infant formulas. Light-emissiveacriflavine compounds that interact with alcohols (e.g., Compound 8) areuseful for product authentication/monitoring testing of neutral anddistilled spirits. Light-emissive chemicals that react with primaryalcohols, aldehydes or ketones (e.g., Compounds 9 and 10) are useful forauthentication or monitoring of neutral spirits, distilled spirits, orsoft drinks.

Selection of Light-emissive Compounds

Light-emissive compounds can be selected, based on one or more of thefollowing properties: (1) a light-emissive compound in the compositionshould interact with an analyte in the product; (2) a light-emissivecompound in the composition should interact with an analyte in theproduct in a concentration dependent manner; (3) a light emissivecompound and the interaction product(s) should be stable and theinteraction should be repeatable; (4) similar lot numbers of the productshould interact the same way with the light-emissive compound; and (5)the light-emissive compound should interact differently with closelyrelated products on the basis of the chemical structures of keyingredients in the product (e.g., to discriminate between brand names ofa product, such as between, for example, Smiroff and Absolut vodkas). Inmany situations, it is desired that multiple light emissive compounds beidentified to authenticate or monitor a single consumer product.

To determine a light-emissive compound that can be used in the analysisof a product, the chemical structure of analytes in the selected productneed not be known, but can be chosen, or assumed to be present in theproduct. At least one analyte is targeted in a particular product. Acandidate light-emissive compound can generally be selected using theguidelines, described above, along with the information listed in Table1 and Table 2. The information listed in the tables is not intended tobe limiting, but provides general information that can be useful in theselection of a light-emissive compound. Table 1 lists reactive groups inlight-emissive compounds that can be useful for identifying particularfunctional groups in a key ingredient of the product. Table 2 listsselected useful initial light emissive compounds. The light emissionfrom a sample containing a light-emissive compound (selected accordingto the guidelines as a result of interactions with analytes in theproduct) can be used to authenticate and monitor products for fraud andquality control.

One preferred method of selection is as follows. A product is selected.The product is diluted until its absorbance is below 0.02. A pluralityof light emissive compounds such as dyes are selected. The dyes arediluted relative to one another so that they all will have an emissionstrength of between 200 and 2000 fluorescence units per ml when 1.4 μl aof dye is added to 1 ml of diluted product (typically between 0.3 and500 micromolar). A series of dilutions of product then are prepared (allbeing below 0.02 absorbance). This, for example, might be dilutions ofproduct such as 1:10, 1:15, 1:20, 1:25 and 1:30. Dyes are added to thesedilutions at 1.4 μl dye/ml diluted product. Dyes also are addedsimilarly to (1) an acceptable, but altered, standard (such as astandard diluted only 5%) and (2) an unacceptable, but altered, standard(such as a standard diluted by >10%) and (3) a nonauthentic but closelyrelated product (such as Peps® where the product is Coca-Cola®). Testsare run in quadruplicate. A fingerprint is generated electronically forthe standard, and an acceptable range which includes the acceptablealtered product but excludes the nonacceptable and nonauthentic productsis A determined electronically, using software and mathematical formulassuch as is described below, pre-selecting the confidence limits (e.g.two standard deviations, three standard deviations, etc.). Multiple dyesare run simultaneously in 96 well plates which are read automatically.Dyes then are selected based upon their ability to distinguish authenticand acceptable products from nonauthentic and nonacceptable products.Further calibration can be carried out. First, a test dilution can beselected as that dilution which is 50% of the lowest dilution of productat which maximum fluorescence for a dye is achieved. Then variables suchas temperature, time of incubation and unacceptable or nonauthenticproducts can be varied, preferably measured in quadruplicate, to permitselection of dyes useful for a given product. As should be understood,using such screening methodology, panels of dyes for producingfingerprint profiles can be selected, without knowledge of thecomposition of the product or the analytes in the product to which thedye binds.

TABLE 1 light-emissive compound reactive group key ingredient functionalgroups activated ester amines or anilines acyl azide amines or anilinesacyl halide amines, anilines, alcohols or phenols acyl nitrile alcoholsor phenols aldehyde amines or anilines alkyl halide amines, anilines,alcohols, phenols or thiols alkyl sulfonate thiols, alcohols or phenolsanhydride alcohols, phenols, amines or anilines aryl halide thiolsaziridine thiols or thioethers carboxylic acid amines, anilines,alcohols or alkyl halides diazoalkane carboxylic acids epoxide thiolshaloacetamide thiols halotriazine amines, anilines or phenols hydrazinealdehydes or ketones hydroxyamine aldehydes or ketones imido esteramines or anilines isocyanate amines or anilines isothiocyanate aminesor anilines

TABLE 2 light-emissive compound analyte acridine orange acid alizarinGarnet R alcohol 9-amino acridine ethanol anthracene ethanol chlorophyllA ethanol/methanol chlorophyll B methanol eosin FAD indole naphthalenealcohol NADPH prolamine protoporphyrin I pryodoxalpyridoamine-5-phosphate quinacrine quinine 6-methoxyquinolinephenanthrene alcohol resorcinol rhodamine 3G (or 6G) riboflavinsalicylic acid serotonin skatole sulfanilic acid sodium salicylate water

Another light-emission tool for product identification is the standardlight emission phenomenon called impurity quenching. Even in dilutesolutions, impurities can cause measurable quenching of light emission.The specific amount of quenching can be exploited to identify a specificlot or batch of a product. See, for example, “Practical Fluorescence,”G. G. Guilbault, Editor, page 32. It is also possible that the lightemission wavelength of the light-emissive compound can shift in thepresence (or absence) of an ingredient in the product. This shift can beused to quantify the amount of ingredient present in the product.

Regional production differences can be determined using two differentmethods. One method involves identifying compounds of regionalspecificity from differences in starting materials. Different suppliersof ingredients in a product will leave different levels of tracecompounds in their supplied materials. Even though these trace compoundsare present at extremely low levels, the light-emissive compounds aresensitive to a level of parts per million and even to parts per billionin some cases. For example, the trace levels of compounds, such asaldehydes and methanol, can be used to identify different varieties(i.e., suppliers) of sucrose and high fructose corn syrup in fruit andcola consumer products. In another example, ethanol distilled from corncontains different trace components than ethanol distilled from canesugar. The identification and analysis of these trace elements can beused to detect product authenticity or detect backfilling (dilution) ofa particular product.

A second method of determining regional differences in a productinvolves analysis of trace elements (or compounds) in, for example, thewater used to dilute the consumer product. The trace elements (orcompounds) can be used as a specific lot number marker. Specifically,levels of calcium, magnesium and/or heavy metals can be used to identifyproducts by “specific lot number water identity.”

Additionally, a company's processes can result in a detectable amount ofat least one other trace material that can identify the companiesspecific product. The identity and quantity of the trace materials makeit possible to identify the lot number of a specific production run. Forexample, many colas have a fixed level of caffeine in the concentrateand in the final product. Light-emissive compounds that indicatecaffeine concentrations can be developed according to methods describedherein.

The relative amounts of key ingredients in a sample can be determined bylight emission analysis. The light emission measurement can be used incombination with other trace light emission analysis to determineauthenticity. For example, vodka must contain 50% ethanol to legally becalled vodka. Additionally, this method can be used to identify a lotnumber or batch number or to determine the authenticity of, for example,orange juice, apple juice, or lemon juice.

The relative amounts of water can be compared in a standard samplestandard and a suspect sample using, for example, the naphthylaminelight-emissive dyes. Sulfonated naphthylamines, such as2-p-toludinylnaphthalene-6-sulfonate (2,6-TNS) and1-anilino-8-naphthalenesulfonate (1,8-ANS), shift light emissionwavelength in water. The relative amount of shift depends on the amountof water in a sample. For example, in water, the spectral sensitivity issubstantially shifted to longer wavelengths, and the light emissionquantum yield and decay times decrease.

Data Analysis

Multi-variant analysis can be used to analyze the light emission resultsof each product sample with each light emissive compound. Typically, theresults are interpreted in comparison to light emission from a standardproduct sample treated in the same way, or a “fingerprint.” All samplescan be analyzed for the presence of key ingredient using alight-emissive compound containing a single light-emissive compound or acombination of light-emissive compounds. The largest and smallest meanvalues are determined for each set of product samples using fourindependent measurements made of the same sample (n=4). The multiplecomparison procedure allows the determination of a critical value (e.g.,at a 95% confidence level) for the difference between the largest andthe smallest sample means, which relates to the differences in therespective products. A difference in the sample means, that is equal toor greater than the critical value, suggests a significant difference inthe products. A significant difference can imply different producttreatments, starting materials and compositions.

Typically, the analysis involves Tukey Multiple Comparison Procedureconducted, e.g., at a 95% confidence level (α0.05). The MultipleComparison Procedure assumes that the number of sample means, k, arebased on independent random samples, each containing the same number ofobservations, n. In this case, s, the standard deviation is the squareroot of the mean square errors (MSE) of the sample means. The MSE has anumber of degrees of freedom, v, associated with it. From k,v, and α,the critical value of the Studentized range, q₆₀ (k,v), can bedetermined (see, for example, Biometrika Tables, Vol. 1, E. L. Pearsonand H. O. Harily, eds., Cambride University Press, Cambridge (1966)). Itthen follows that the distance, omega (T) is$\omega = {{q_{\alpha}( {k,v} )}\quad \frac{s}{N^{0.5}}}$

Tukey analysis can allow the identification of sample means that do notmatch the standard products. If two measurements differ by a valuegreater than omega, then the two samples are different. If not, thesamples are pairs have substantially similar compositions (i.e., are thesame composition, but could be different batches). Each light-emissivecompound/product sample system can be considered a single variant.Combining the analyses for each of the light-emissive compounds togethercan lead to a multi-variant analysis program that we have developed asoftware program for. That this multi-variant light-emissive productauthenticity analysis can be carried out using, for example,spread-sheet type computer programs.

While Tukey analysis has been described herein, it is to be appreciatedthat other multi-variant methods of analysis may be used. Such alternatemethods include, for example, Duncan's multiple range analysis andNewman-Kuls analysis, as described in Biostatistical Analysis, 3rdEdition, J. H. Zar, Prentice-I-lall, Upper Saddle River, N.J. (1996).

FIG. 2 is a data flow diagram representing the overall processing inthis system of the invention. Standard samples are processed withselected compounds by first processing system 40 to produce lightemission results 42. It is possible that these light emission resultscould be stored in a database 44. Similarly, unknown samples areprocessed by a processing system 46 using the same selected compounds.The processing system 46 produces light emission results 48, i.e., afingerprint for each unknown sample. A comparison procedure is performedby a comparator 50 to produce an indication of the authenticity of thesample. The comparison can be performed using the Tukey multiplecomparison procedure described above. This computer may receive the datafrom the light emission results from processing systems 40 and 46 eitherdirectly from those systems or over a computer network. The comparatoralso may receive the fingerprint of the standard samples from a databasewhich may be either local to or remote from the computer running thecomparison procedure.

A suitable computer system to implement the comparator 50 typicallyincludes a main unit connected to an output device, such as a display,and an input device, such as a keyboard. The main unit generallyincludes a processor connected to a memory system via an interconnectionmechanism. The input device is also connected to the processor andmemory system via the connection mechanism, as is the output device.

It should be understood that one or more output devices may be connectedto the computer system. Example output devices include a cathode raytube (CRT) display, liquid crystal displays (LCD), printers,communication devices such as a modem, and audio output. It should alsobe understood that one or more input devices may be connected to thecomputer system. Example input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication device, audio input andscanner. It should be understood the invention is not limited to theparticular input or output devices used in combination with the computersystem or to those described herein.

The computer system may be a general purpose computer system which isprogrammable using a high level computer programming language, such as“C”, or “Pascal”. The computer system may also be specially programmed,special purpose hardware. In a general purpose computer system, theprocessor is typically a commercially available processor, of which theseries x86 processors, available from Intel, and the 680X0 seriesmicroprocessors available from Motorola are examples. Many otherprocessors are available. Such a microprocessor executes a programcalled an operating system, of which UNIX, DOS and VMS are examples,which controls the execution of other computer programs and providesscheduling, debugging, input/output control, accounting, compilation,storage assignment, data management and memory management, andcommunication control and related services. The processor and operatingsystem define a computer platform for which application programs inhigh-level programming languages are written.

A memory system typically includes a computer readable and writeablenonvolatile recording medium, of which a magnetic disk, a flash memoryand tape are examples. The disk may be removable, known as a floppy diskor an optical disk, or permanent, known as a hard drive. A disk has anumber of tracks in which signals are stored, typically in binary form,i.e., a form interpreted as a sequence of one and zeros. Such signalsmay define an application program to be executed by the microprocessor,or information stored on the disk to be processed by the applicationprogram. Typically, in operation, the processor causes data to be readfrom the nonvolatile recording medium into an integrated circuit memoryelement, which is typically a volatile, random access memory such as adynamic random access memory (DRAM) or static memory (SRAM). Theintegrated circuit memory element allows for faster access to theinformation by the processor than does the disk. The processor generallymanipulates the data within the integrated circuit memory and thencopies the data to the disk when processing is completed. A variety ofmechanisms are known for managing data movement between the disk and theintegrated circuit memory element, and the invention is not limitedthereto. It should also be understood that the invention is not limitedto a particular memory system.

It should be understood the invention is not limited to a particularcomputer platform, particular processor, or particular high-levelprogramming language. Additionally, the computer system may be amultiprocessor computer system or may include multiple computersconnected over a computer network.

Materials and Methods

The methods were developed to optimize analysis or determine theauthenticity or tampering of a product in the water and/or organiccomponent of the product. The general methods for using Compounds 1-10are generally described below.

A Beckman Biomek 1000 automated workstation (Beckrnan Instruments,Columbia, MD) was used to make dilutions and place 150 microliters ofthe light-emissive compound into a test plate, although any automateddispensing workstation can be used. The test plate can be made from anysuitable material and can have any number of wells, such as 6, 24, 96 or384 wells (Corning-Costar, Falcon-Collaborative, microwell test plates).The light emission of the light emissive compound in the absence ofproduct is Fp, and the light emission after exposing the light-emissivecompound to the product is Fd. The Fd and Fp light emission analysis forthe purpose of these experiments was made using a Molecular DynamicsFluorlmager 575, but any microplate reader can be used (e.g.,Cytofluor). Bandpass and cutoff filters are used to isolate excitationwavelengths from emission spectra due to light emission from the sample.Fd light emission analysis was made for each chemical in each well ofthe test plate. Repetition of measurements allows correction forsystematic variability due, for example, to automatic pipetting (<5%).Next, 150 microliters of product are added to the chemicals in themicrowells using the Beckman Biomek 1000 automated workstation. Thechemical and the product are allowed to react for a period of time andtemperature that is specific for each product and chemical. Change inchemical light emission due to the presence of the product is determinedby calculation using the equation [(Fd−Fp)/Fd]×100.

In certain embodiments, an immutable standard, such as a ruby or otherprecious stone, may be used to compensate for variations in the laseroutput signal intensity. For such embodiments, the immutable standardcan be placed in one of the wells of the test plate.

Compound 1,5-(2-carbohydrzinomethylthioacetyl)aminofluorescein, wasobtained from Molecular Probes, Inc., Eugene, Oreg., Lot 2841-1. Thefinal concentration of the working solution can range between 0.5 and 10micromolar. Compound 1 has an excitation maximum at 488 nm at neutral pHand 356 nm at pH 8. Compound 1 has an emission maximum at 520 nm. See,R. E. Hileman, et al., Bioconjugate Chem. 5:436 (1994) for the synthesisof the compound.

Compound 2 and Compound 3 should be used in the method together.Compound 2, 5-(4,6-dicillorotriazinyl)aminofluorescein, was obtainedfrom Molecular Probes, Inc., Eugene, Oreg., Lot 2851-1. A stock solutionof Compound 2 was prepared in dimethyl sulfoxide (DMSO, ACS reagent,Sigma Chemical, St Louis, Mo.). The final concentration of the workingsolution can range between 0.5 and 10 micromolar. Compound 2 has anexcitation maximum at 495.7 nm and emission maximum at 516.3 nm. For areference that describes the original use of this compound, see, Barskiiet al., Izv. Akad. Nuak SSSR, V. E. (1968) PN 101.

Compound 3, Fluo-3, pentaammonium salt, was obtained from MolecularProbes, Inc., Eugene, Oreg., Lot 2641-6. The final concentration of theworking solution can range between 0.5 and 10 micromolar. Compound 3 hasan excitation maximum at 510 nm and emission maximum at 530 nm. Fluo-3was developed for measuring calcium levels in cellular experiments. See,for example, Tsien, R., et al., J. Biol. Chem. 264:8171 (1989).

Compound 4, proflavine hemisulfate (3,6-diaminoacridine hemisulfate) wasobtained from Sigma-Aldrich, St. Louis, Mo. The final concentration ofthe working solution can range between 0.5 and 10 micromolar. Compound 4has an emission maximum at 515 nm in methanol. Proflavine was developedas a fabric dye and for cell staining procedures. See, for example,Chan, L. M., et al., Biochem. Biophys. Acta, 204:252 (1970).

Compound 5, tetra(tetramethylammonium) salt, was obtained from MolecularProbes, Inc., Eugene Oreg. The final concentration of the workingsolution can range between 0.5 and 20 micromolar, depending on theproduct tested. Compound 5 has an excitation maximum at 488 nm and anemission maximum around 535 nm. Compound 5 was developed at MolecularProbes as Sodium Greenlm for the fluorometric determination of Na+concentrations.

Compound 6, acridine orange hydrochloride hydrate, obtained fromSigma-Aldrich, St. Louis, Mo. The final concentration of the workingsolution can range between 0.5 and 20 micromolar. Compound 6 has anexcitation maximum at approximately 490 nm and emission maximum at 519nm. Compound 6 can be used for printing inks and as a stain for fats andlipids in biological samples. See, for example, Clark, G., “StainingProcedures'” ed. Williams and Wilkins, Baltimore 1981 pp. 48, 57, 61,71, 72, 86, 87, 89, 90, and 429.

Compound 7, BTC-5N (Costlei et al., J. of Chem. Society Perkinstranslation 2, p. 1615), was obtained from Molecular Probes, Inc.,Eugene, Oreg. The final concentration of the working solution can rangebetween 0.5 and 20 micromolar. Compound 7 has an excitation maximum atapproximately 415 nm and an emission maximum at 515 nm.

Compound 8, acriflavine, is composed of an approximate 8 to 1 mixture of3,6-diamino- 10-methylacridinium chloride and 3,6-diaminoacridine, andwas obtained from Sigma-Aldrich, St.-Louis, Mo. The working solutionconcentration can range between 0.5 and 20 micromolar, depending on theproduct tested. Compound 8, in its neutral form, has an excitationmaximum in ethanol at 483 nm and an emission maximum at 517 nm with along-lasting emission state that can be used to identify the relativelevels of ethanol in a sample. The long-lasting emission in ethanol isnoted by Furumoto, H. W. and Ceccon, H. L., IEEE J. Quantum Electron.,QE-6, 262, (1970). Compound 8 is an ordinary biological stain and isuseful as a light-emissive compound and a Schiff reagent. See, forexample, “Conn's, Biological Stains,” 9th ed.: Lillie, R. D., Ed.;Williams and 25 Wilkins: Baltimore, 1977; p. 355.

Compound 9, 4-aminofluorescein, was obtained from Sigma-Aldrich, St.Louis, Mo. The working solution concentration can range between 0.5 and20 micromolar, depending on the product tested. Compound 9 has anexcitation maximum at 496 nm and an emission maximum at 530 nm. See, forexample, Coons, A. H., et al., J.,Exp. Med. 91:1-14 (1950).

Compound 10, 5-aminofluorescein, obtained from Sigma-Aldrich, St. Louis,Mo., was used in a similar manner and at similar concentrations asCompound 9. The emission is at 530 nm. Glabe et al, Anal. Biochem,130:287-294 (1983).

Compound 11, sulfite blue coumarin, S-6902, was obtained from MolecularProbes, Eugene, Oreg. Compound 11 has an excitation maximum at 325 rmand an emission maximum at 373 nm. Compound 11 can be useful formeasuring sulfites. Sulfite contamination in high fructose corn syrup isa problem well known in the corn processing and milling industry.

Compound 12, courmarin diacid cryptand (CD222) (Costlei et al., J. ofChem. Society Perkins translation 2, p.1615), was obtained fromMolecular Probes, Eugene, Oerg. Compound 12 is a ratio dye with anexcitation maximum at 365 nm and emission maximum at 465 nm. Compound 12is a potassium sensitive dye, enabling authentication based potassiumbenzoate, a preservative in many cola drinks.

Compound 13, Eosin Y, was obtained from SigmaAldrich, certified Grade,St. Louis, Mo. Compound 13 has an excitation maximum at 522 nm and anemission maximum at 551 nm. Compound 13 is a pH-sensitive light-emissivecompound.

EXAMPLE 1

Neutral Spirits

The analysis methods of the invention can be used in the wine anddistilled spirits industry to determine product authenticity, defendinternational trademarks, document product quality, and detect productbackfilling (i.e., dilution with lower quality ingredients). In thisindustry, the origin and source of the ethanol in a product can be usedto determine product authenticity. The product label must correctlyrepresent the contents in a manufacturer's bottle. Previously, there wasno practical method for determining the source of ethanol or neutralspirits (96% ethanol).

A double blind experiment was conducted to determine the differencesbetween 6 neutral spirits samples. In addition, if there wereduplicates, the experiment was designed to identify the duplicates.

The neutral spirits product origins can be identified from the datapresented in Table 3 and Table 4. Referring to Table 3, the level oflight emission upon excitation was monitored in an array of six samples(10-1, 10-2, 10-3, 10-4, 10-5, and 10-6) that were each tested fourtimes (A, B, C, and D) with a pair of light-emissive compounds. Withineach set, each sample of the product was tested four times with alight-emissive compound. The excitation wavelength was 522 nm. Thelight-emissive compounds were Compound 2 and Compound 3.

Stock solutions of the light-emissive compounds were prepared bydissolving Compound 2 in DMSO at a concentration of 2 mM and Compound 3in DMSO at a concentration of 1 mM.

The concentrations of the working solutions of light-emissive compoundswere optimized against known samples of neutral spirits. The optimumconcentrations were determined from the concentrations of light-emissivecompounds that provides emission intensities that are capable ofdiscriminating known neutral spirits samples from other samples by avalue greater than omega. The working solution of Compound 2 wasprepared by diluting 120 μL of the stock solution dye in 20 mL ofdistilled water. The working solution of Compound 3 was prepared bydiluting 100 μL of the stock solution in 20 mL of distilled water.

Both Compound 2 and Compound 3 require a 53OBP+15 nm band pass filter toreduce the excitation wavelength intensity during the emissionmeasurements. The intensity of the emission was measured in relativefluorescence units

TABLE 3 A 10-1 A 10-2 A 10-3 A 10-4 A 10-5 A 10-6 B 10-1 B 10-2Measurement 1 0.2276 −0.5481 −0.2021 −0.2030 −0.5479 0.1773 0.2088−0.5474 Measurement 2 0.2299 −0.5454 −0.1991 −0.1979 −0.5354 0.20960.2363 −0.5417 Measurement 3 0.2384 −0.5471 −0.2095 −0.1966 −0.54790.1811 0.2148 −0.5524 Measurement 4 0.2468 −0.5573 −0.1982 −0.1939−0.5434 0.1932 0.2113 −0.5548 Variance: 7.618E − 05 2.866E − 05 2.629E −05 1.467E − 05 3.466E − 05 2.119E − 03 1.584E − 04 3.377E − 05 Mean:0.2357 −0.5495 −0.2022 −0.1978 −0.5437 0.1903 0.2178 −0.5491 B 10-3 B10-4 B 10-5 B 10-6 C 10-1 C 10-2 C 10-3 C 10-4 Measurement 1 −0.2395−0.2079 −0.5699 0.1670 0.2480 −0.5471 −0.1908 −0.1828 Measurement 2−0.2116 −0.2309 −0.5627 0.2337 0.2683 −0.5383 −0.1899 −0.1886Measurement 3 −0.2424 −0.2381 −0.5637 0.1677 0.2759 −0.5330 −0.1883−0.1862 Measurement 4 −0.2312 −0.2292 −0.5769 0.1810 0.2501 −0.5356−0.1911 −0.1756 Variance: 1.936E − 04 1.689E − 04 4.32E − 05 9.967E − 041.876E − 04 3.765E − 05 1.506E − 06 3.184E − 05 Mean: −0.2312 −0.2265−0.5683 0.1874 0.2606 −0.5385 −0.1900 −0.1833 C 10-5 C 10-6 D 10-1 D10-2 D 10-2 D 10-4 D 10-5 D 10-6 Measurement 1 −0.5449 0.2154 0.2435−0.5597 −0.2321 −0.2200 −0.5633 0.1974 Measurement 2 −0.5615 0.22340.2609 −0.5486 −0.2191 −0.2104 −0.5626 0.2431 Measurement 3 −0.54880.2247 0.2687 −0.5496 −0.1994 −0.1999 −0.5552 0.2597 Measurement 4−0.5453 0.2428 0.2861 −0.5452 −0.2055 −0.1891 −0.5466 0.2766 Variance:6.044E − 05 1.341E − 04 3.124E − 04 3.882E − 05 2.134E − 04 1.776E − 046.078E − 05 1.161E − 03 Mean: −0.5501 0.2266 0.2648 −0.5508 −0.2141−0.2048 −0.5570 0.2442 MSE = OMEGA = 0.0001835 0.0354918

TABLE 4 Fingerprint Data A 10-1 A 10-2 A 10-3 A 10-4 A 10-5 A 10-6 B10-1 B 10-2 B 10-3 B 10-4 B 10-5 B 10-6 A 10-1 0 1 1 1 1 1 0 1 1 1 1 1 A10-2 1 0 1 1 0 1 1 0 1 1 0 1 A 10-3 1 1 0 0 1 1 1 1 0 0 1 1 A 10-4 1 1 00 1 1 1 1 0 0 1 1 A 10-5 1 0 1 1 0 1 1 0 1 1 0 1 A 10-6 1 1 1 1 1 0 0 11 1 1 0 B 10-1 0 1 1 1 1 0 0 1 1 1 1 0 B 10-2 1 0 1 1 0 1 1 0 1 1 0 1 B10-3 1 1 0 0 1 1 1 1 0 0 1 1 B 10-4 1 1 0 0 1 1 1 1 0 0 1 1 B 10-5 1 0 11 0 1 1 0 1 1 0 1 B 10-6 1 1 1 1 1 0 0 1 1 1 1 0 C 10-1 0 1 1 1 1 1 1 11 1 1 1 C 10-2 1 0 1 1 0 1 1 0 1 1 0 1 C 10-3 1 1 0 0 1 1 1 1 1 1 1 1 C10-4 1 1 0 0 1 1 1 1 1 1 1 1 C 10-5 1 0 1 1 0 1 1 0 1 1 0 1 C 10-6 0 1 11 1 1 0 1 1 1 1 1 D 10-1 0 1 1 1 1 1 1 1 1 1 1 1 D 10-2 1 0 1 1 0 1 1 01 1 0 1 D 10-3 1 1 0 0 1 1 1 1 0 0 1 1 D 10-4 1 1 0 0 1 1 1 1 0 0 1 1 D10-5 1 0 1 1 0 1 1 0 1 1 0 1 D 10-6 0 1 1 1 1 1 0 1 1 1 1 1 C 10-1 C10-2 C 10-3 C 10-4 C 10-5 C 10-6 D 10-1 D 10-2 D 10-3 D 10-4 D 10-5 D10-6 A 10-1 0 1 1 1 1 0 0 1 1 1 1 0 A 10-2 1 0 1 1 0 1 1 0 1 1 0 1 A10-3 1 1 0 0 1 1 1 1 0 0 1 1 A 10-4 1 1 0 0 1 1 1 1 0 0 1 1 A 10-5 1 0 11 0 1 1 0 1 1 0 1 A 10-6 1 1 1 1 1 1 1 1 1 1 1 1 B 10-1 1 1 1 1 1 0 1 11 1 1 0 B 10-2 1 0 1 1 0 1 1 0 1 1 0 1 B 10-3 1 1 1 1 1 1 1 1 0 0 1 1 B10-4 1 1 1 1 1 1 1 1 0 0 1 1 B 10-5 1 0 1 1 0 1 1 0 1 1 0 1 B 10-6 1 1 11 1 1 1 1 1 1 1 1 C 10-1 0 1 1 1 1 0 0 1 1 1 1 0 C 10-2 1 0 1 1 0 1 1 01 1 0 1 C 10-3 1 1 0 0 1 1 1 1 0 0 1 1 C 10-4 1 1 0 0 1 1 1 1 0 0 1 1 C10-5 1 0 1 1 0 1 1 0 1 1 0 1 C 10-6 0 1 1 1 1 0 1 1 1 1 1 0 D 10-1 0 1 11 1 1 0 1 1 1 1 0 D 10-2 1 0 1 1 0 1 1 0 1 1 0 1 D 10-3 1 1 0 0 1 1 1 10 0 1 1 D 10-4 1 1 0 0 1 1 1 1 0 0 1 1 D 10-5 1 0 1 1 0 1 1 0 1 1 0 1 D10-6 0 1 1 1 1 0 0 1 1 1 1 0

(rfu). The emission measurements are always made in the region of linearresponse, which on this fluorescence measuring instrument is madebetween 200 and 2000 rfu.

Each working solution (150 μL) was added to the test plate using anautomated handling device with less than 5% error in volume measurement.The working solution/plate combination was measured for backgroundfluorescence to account for variability in composition, platedimensions, and laser output. Excitation and emission experiments can berun on any laser or non-laser fluorescence detection system. In this setof experiments the measurements were made using a Fluorlmager 575(Molecular Dynamics, Sunnyvale, Calif.).

The neutral spirits samples were added directly to the sample plate. Akey discovery in analyzing neutral spirits (96% ethanol) is that theanalysis of the residual water is important. The signal from Compound 3is designed to analyze the residual water. However, the highconcentrations of ethanol in the samples masks the signal from thewater. For this identification method to work well, the ethanol isremoved under vacuum from the samples after they have been added to theindividual microwells of the plate. This reduction allows the exactanalysis of the water in the neutral spirits samples. Since thereduction takes place directly on the microwell plates, all samples aretreated equally and the process is automated by placing a vacuum bell onthe automated plate-handling work station.

The results of the experiment are presented in Table 3. Variance andmean were calculated for each group (A, B, C, or D) of 4 measurements.The 95% confidence levels were used for this fingerprint analysis. Iftwo sample means differ by an amount greater than the omega, the samplesare different (i.e., substantially different in composition). Forexample, in test A, sample 10-1 had a mean light emission intensity of0.2357 and sample 10-2 had a mean light emission intensity of −0.5495.The difference in light emission intensity was 0.7852. The omega fortest A was 0.0354918. If the difference (0.7852) is greater than omega(0.0354918) for any two samples, then the samples are different.Therefore, 10-1 and 10-2 are different. The comparison is made basedstrictly on the statistical data and can be done automatically, withoutthe need for further interpretation.

The fingerprint data are presented in Table 4 to make all possiblecomparisons. A value of 1 in Table 4 indicates that the two sample meansdiffer by more than omega. The value of 0 indicates that two samples donot differ by more than omega. Thus, a value of 0 signifies that thesamples are pairs (i.e., substantially similar in composition, such asdifferent batches or lots) or that the sample tested against itself(along the upper left-to-lower right diagonal of Table 4) and a value ofI signifies that the samples are different. When sample pairs areconsistently different, the samples are determined to have substantiallydifferent compositions (i.e., different brands altogether). As a resultof the fingerprinting analysis in Table 4, products 10-1 and 10-6, 10-2and 10-5, and 10-3 and 10-4 were pairs (i.e., substantially similar incomposition) that are different from each other (i.e.,different lots).

EXAMPLE 2

Distilled Spirits

In a manner similar to that described in Example 1, it is possible toauthenticate distilled spirits, such as vodka. For this fingerprintanalysis, Compound 1, Compound 2, Compound 3, Compound 4, Compound 5,Compound 6, Compound 7, and Compound 8 can be used.

The stock solution of Compound 1 was 1.5 mM in a 1:1 DMSO/water mixture.The stock solution of Compound 2 was 2 mM in DMSO. The stock solution ofCompound 3 was 0.5 mM in a 1:1 DMSO/water mixture. The stock solution ofCompound 4 was 1 mM in DMSO. The stock solution of Compound 5 was 1 mMin distilled water. The stock solution of Compound 6 was 1 mM in DMSO.The stock solution of Compound 7 was 1 mM in distilled water. The stocksolution of Compound 8 was 4 mg/mL in ethanol (chromatography grade,Sigma Chemical Company, St. Louis, Mo.).

Working solution concentrations were determined as in Example 1. Theoptimum concentration of light-emissive compound was determined to bethe level that allows discrimination of known samples having valuedifferences greater than omega. The working solution of Compound 2 wasprepared by diluting 120 μL of the 2 mM stock solution in 20 mL ofdistilled water. The working solution of Compound 3 was prepared bydiluting 100 μL of the stock solution in 20 mL of distilled water. Theworking solution of Compound 4 was prepared by diluting 100 μL of thestock solution in 50 mL of distilled water. The working solution ofCompound 5 was prepared by diluting 75 μL of the stock solution in 50 mLof distilled water. The working solution of Compound 6 was prepared bydiluting 50 mL of the stock solution in 50 mL of ethanol. The workingsolution of Compound 7 was prepared by diluting 25 μL of the stocksolution in 50 mL of distilled water. The working solution of Compound 8was prepared by diluting 50 μL of the stock solution in 50 mL ofdistilled water. Compounds 1, 2, 3, 4, 5, 6, and 8 require a 53OBP+15 rmband pass filter to reduce the excitation wavelength intensity duringemission measurements. Compound 7 requires the use of a 515 nm Long Passfilter (LP).

Each working solution (150 μL) was added to the test plate using anautomated handling device with less than 5% error in volume measurementas in Example 1. The distilled spirits (vodka) samples were analyzed asdescribed in Example 1.

EXAMPLE 3

Carbonated Drinks and Fruit Beverages

The analysis methods of the invention can be used in the soft drink andfruit juice industry, particularly to check third party re-formulationsin every lot to monitor licensing agreements, for example. The analysisspeed needed to check samples of this type should be faster than 300samples/hour. This was not a double blind test.

Product formulations can be verified by methods similar to thosedescribed in Example 1. The fingerprint is the same when the product isproduced to the same high quality of standards. Referring to Table 5,light emission was monitored in an array of six samples (Pepsi 1, Pepsi2, Diet Pepsi 3, Coke Classic 4, Diet Coke 5, and Black Cherry 6) thatwere each tested four times with the four different light-emissivecompounds (A, B, C, and D). Test A used compound 1. Test B used Compound2. Test C used Compound 3. Test D used Compound 9. Each sample of theproduct was tested four times with each light-emissive compound.

The test methods were generally conducted in the following manner. Thebeverages or juices were diluted 1:10 to 1:300 with water for optimumreaction with the light emissive compounds. The optimum response of thesample is determined empirically, by using a concentration curve tomaximize emission response. The sample concentration was selected togive one-half of the maximum emission response with the tested sample.

TABLE 5 A A A A A A Diet Coke Diet Black B B Pepsi 1 Pepsi 2 PepsiClassic Coke Cherry Pepsi 1 Pepsi 2 Measurement 1 −0.8311 −0.8360−0.7252 −0.8701 −0.6664 −0.8449 −0.3578 −0.3040 Measurement 2 −0.8473−0.8358 −0.7179 −0.8716 −0.6614 −0.8368 −0.3471 −0.3294 Measurement 3−0.8315 −0.8349 −0.7157 −0.8721 −0.6414 −0.8432 −0.3254 −0.3186Measurement 4 −0.8407 −0.8293 −0.7145 −0.8633 −0.6544 −0.8368 −0.3368−0.2848 Variance: 6.105E − 05 9.985E − 06 2.301E − 05 1.644E − 05 1.179E− 04 1.795E − 05 1.924E − 04 3.725E − 04 Mean: −0.8376 −0.8340 −0.7183−0.8693 −0.6559 −0.8404 −0.3418 −0.3092 B B B B C C Diet Coke Diet BlackC C Diet Coke Pepsi Classic Coke Cherry Pepsi 1 Pepsi 2 Pepsi ClassicMeasurement 1 0.3592 −0.2481 0.7057 −0.6725 2.3477 2.4311 3.2869 2.2283Measurement 2 0.3953 −0.2200 0.7018 −0.6583 2.4218 2.2661 3.2057 2.2739Measurement 3 0.3907 −0.2192 0.7283 −0.6620 2.4042 2.4579 3.2358 2.3609Measurement 4 0.4028 −0.2119 0.7634 −0.6731 2.4532 2.5020 3.3130 2.4228Variance: 3.886E − 04 2.551E − 02 7.983E − 04 5.589E − 05 1.958E − 031.061E − 02 2.357E − 05 7.589E − 03 Mean: 0.3870 −0.2248 0.7248 −0.66652.4067 2.4143 3.2603 2.3215 C C D D D D Diet Black D D Diet Coke DietBlack Coke Cherry Pepsi 1 Pepsi 2 Pepsi Classic Coke Cherry Measurement1 3.4794 1.6844 8.6589 8.8383 7.3358 8.0844 6.8748 10.4029 Measurement 23.4689 1.6986 8.7641 9.1611 7.3981 8.3034 6.9779 10.5467 Measurement 33.5929 1.7489 9.1029 9.1162 7.5821 8.2324 7.0303 10.5861 Measurement 43.6457 1.9201 9.4517 9.3065 7.5014 8.6027 7.1879 11.0469 Variance:7.507E − 03 1.174E − 02 0.1288 3.833E − 02 1.192E − 02 4.752E − 021.705E − 02 7.776E − 02 Mean: 3.5467 1.7630 8.9944 9.1055 7.4544 8.30577.0177 10.6456 MSE = OMEGA = 0.0152267 0.3232988

TABLE 6 Fingerprint Data A A A Diet A Diet A Blk B B B Diet B Diet B BlkPepsi 1 Pepsi 2 Pepsi A Coke Coke Che Pepsi 1 Pepsi 2 Pepsi B Coke CokeChe A Pepsi 1 0 0 0 0 0 0 1 1 1 1 1 0 A Pepsi 2 0 0 0 0 0 0 1 1 1 1 1 0A Diet Pepsi 0 0 0 0 0 0 1 1 1 1 1 0 A Coke 0 0 0 0 0 0 1 1 1 1 1 0 ADiet Coke 0 0 0 0 0 0 0 1 1 1 1 0 A Blk Che 0 0 0 0 0 0 1 1 1 1 1 0 BPepsi 1 1 1 1 1 1 1 0 0 1 1 1 1 B Pepsi 2 1 1 1 1 1 1 0 0 1 1 1 1 B DietPepsi 1 1 1 1 1 1 1 1 0 1 1 1 B Coke 1 1 1 1 1 1 0 0 1 0 1 1 B Diet Coke1 1 1 1 1 1 1 1 1 1 0 1 B Blk Che 0 0 0 0 0 0 1 1 1 1 1 0 C Pepsi 1 1 11 1 1 1 1 1 1 1 1 1 C Pepsi 2 1 1 1 1 1 1 1 1 1 1 1 1 C Diet Pepsi 1 1 11 1 1 1 1 1 1 1 1 C Coke 1 1 1 1 1 1 1 1 1 1 1 1 C Diet Coke 1 1 1 1 1 11 1 1 1 1 1 C Blk Che 1 1 1 1 1 1 1 1 1 1 1 1 D Pepsi 1 1 1 1 1 1 1 1 11 1 1 1 D Pepsi 2 1 1 1 1 1 1 1 1 1 1 1 1 D Diet Pepsi 1 1 1 1 1 1 1 1 11 1 1 D Coke 1 1 1 1 1 1 1 1 1 1 1 1 D Diet Coke 1 1 1 1 1 1 1 1 1 1 1 1D Blk Che 1 1 1 1 1 1 1 1 1 1 1 1 HFCS/Sucrose Liquids C C C Diet C DietC Blk D D D Diet D Diet D Blk Pepsi 1 Pepsi 2 Pepsi C Coke Coke ChePepsi 1 Pepsi 2 Pepsi D Coke Coke Che A Pepsi 1 1 1 1 1 1 1 1 1 1 1 1 1A Pepsi 2 1 1 1 1 1 1 1 1 1 1 1 1 A Diet Pepsi 1 1 1 1 1 1 1 1 1 1 1 1 ACoke 1 1 1 1 1 1 1 1 1 1 1 1 A Diet Coke 1 1 1 1 1 1 1 1 1 1 1 1 A BlkChe 1 1 1 1 1 1 1 1 1 1 1 1 B Pepsi 1 1 1 1 1 1 1 1 1 1 1 1 1 B Pepsi 21 1 1 1 1 1 1 1 1 1 1 1 B Diet Pepsi 1 1 1 1 1 1 1 1 1 1 1 1 B Coke 1 11 1 1 1 1 1 1 1 1 1 B Diet Coke 1 1 1 1 1 1 1 1 1 1 1 1 B Blk Che 1 1 11 1 1 1 1 1 1 1 1 C Pepsi 1 0 0 1 0 1 1 1 1 1 1 1 1 C Pepsi 2 0 0 1 0 11 1 1 1 1 1 1 C Diet Pepsi 1 1 0 1 0 1 1 1 1 1 1 1 C Coke 0 0 1 0 1 1 11 1 1 1 C Diet Coke 1 1 0 1 0 1 1 1 1 1 1 1 C Blk Che 1 1 1 1 1 0 1 1 11 1 1 D Pepsi 1 1 1 1 1 1 1 0 0 1 1 1 1 D Pepsi 2 1 1 1 1 1 1 0 0 1 1 11 D Diet Pepsi 1 1 1 1 1 1 1 1 0 1 1 1 D Coke 1 1 1 1 1 1 1 1 1 0 1 1 DDiet Coke 1 1 1 1 1 1 1 1 1 1 0 1 D Blk Che 1 1 1 1 1 1 1 1 1 1 1 0

Stock solutions of the light-emissive compounds were prepared bydissolving Compound 1 at a concentration of 1.5 mM in 1:2 DMSO/water,Compound 2 at a concentration of 2 mM in DMSO, Compound 3 at aconcentration of 1 mM in DMSO, and Compound 9 at a concentration of 10mM in DMSO.

The concentrations of working solutions of light emissive compounds wereoptimized as described in Example 1. Optimum concentrations werecalculated from the concentrations of light-emissive compound thatprovide emission intensity values that can discriminate a standardproduct sample from other the unknown samples by a value greater thanomega. The working solution of Compound I was prepared by diluting 75 μLof the stock solution in 50 mL of distilled water. The working solutionof Compound 2 was prepared by diluting 120 μL of the stock solution in20 mL of distilled water. The working solution of compound 3 wasprepared by diluting 100 μL of the stock solution in 20 mL of distilledwater. The working solution of Compound 9 was prepared by diluting 50 μLof the stock solution in 50 mL of distilled water.

Compounds 1, 2, 3, and 9 require a 530BP+15 nm band pass filter toreduce the excitation wavelength intensity during the emissionmeasurements. The sample placement and emission analysis was carried outas described in Example 1. The results of the experiment are presentedin Table 5. There were four different measurements (A, B, C, and D) madefor each sample in combination with each light-emissive compound. Eachmeasurement was repeated four times to demonstrate the level ofreproducibility. Variance and mean were calculated for each group of 4measurements. The 95% confidence levels were used for this fingerprintanalysis. If two sample means differ by an amount greater than omega,the samples are different (i.e. substantially similar in composition).For example, in the test with light-emissive compound D, sample Pepsi 1had a mean light emission of 8.9944 and sample Pepsi 2 had a mean lightemission of 9.1055. The difference in light emission was 0.1111. Theomega for the test was 0.3232988. If the difference (0.1111) is greaterthan omega for any two samples, then the samples are substantially thesame. Therefore, Pepsi 1 and Pepsi 2 are substantially the same.

The fingerprint data are presented in Table 6 to make all possiblecomparisons and are established in the same manner as in Example 1. As aresult of the fingerprinting analysis in Table 6, Pepsi 1 and Pepsi 2are pairs, with similar compositions, and the other samples are notrelated.

EXAMPLE 4

One-step Analysis of Beverages

The methods of Example 3 can be modified to monitor key ingredients inbeverages. Importantly, it was discovered that the key ingredients thatare currently measured by standard analytical methods for authenticitymonitoring can also be measured by the methods of automated emissionmeasurements of the invention. In other words, there are someingredients inherent to certain products that have characteristiclight-emission properties. The methods can be used to analyze thesecomponents in a single-plate analysis with the all the light-emissivecompounds combined together, thereby allowing modification andautomation of the method into a simple, one-step inexpensive emissionscan.

Standard ingredients that are monitored in colas are, for example, highfructose corn syrup (HFCS)I caffeine, potassium benzoate, sodiumbenzoate, pH, and aspartame. Two combinations of light-emissiveCompounds have been developed for the analysis of colas.

Combination 1 allows monitoring of sugar (or HFCS) sources, caffeine,pH, and preservatives (such as potassium or sodium benzoate) combination1 is useful for analyzing ordinary carbonated beverages. Combination 2is tailored for the analysis of diet carbonated beverages and allowsmonitoring of aspartame, caffeine, pH, and preservatives. TheCombinations are designed to detect changes in specific ingredients fromat 0.1, 0.3, 0.5, 1, 2, and 3 percent reduction levels.

Combination 1 includes Compound 1, Compound 3, and Compound 11 for theanalysis of sugar (or HFCS). Caffeine is a light-emissive compound aloneand does not require addition of another component to the mixture. Thecommon preservatives, potassium and sodium benzoate, can be identifiedusing a number of light-emissive compounds. For example, Compound 12 isa potassium sensitive dye. The carboxylic acid on the benzoate group isreactive with all alkyl halide, carbodimide, and alcohol containinglight-emissive compounds (see Table 1). The pH can be determined usingany pH-sensitive light-emissive compound that emits in range from of pHfrom 1-4 (most soft drinks range in pH from 2.4-4.0). A specific exampleof a pH-sensitive light emissive compound is Compound 13.

Stock solutions of the light-emissive compounds were prepared bydissolving Compound 1 at a concentration of 1.5 mM in a 1:1 DMSO/watermixture, Compound 3 at a concentration of 0.5 mM in 1:1 mixture ofDMSO/water, Compound 11 at a concentration of 10 mM in ethanol, Compound12 at a concentration of 10 mM in DMSO, and Compound 13 at aconcentration of 10 mM in distilled water.

The working concentrations were optimized for identification of keyingredients for each soft drink beverage product, as described inExample 1. The working solution of Compound 1 was prepared by diluting75 μL of the stock solution in 50 mL of distilled water. The workingsolution of Compound 3 was prepared by diluting 100 μL of the stocksolution in 20 mL of water. The working solution of Compound 11 wasprepared by diluting 50 μL of the stock solution in 50 mL of distilledwater. The working solution of Compound 12 was prepared by diluting 50μL of the stock solution in 50 mL of distilled water. The workingsolution of Compound 13 was prepared by diluting 50 μL in 100 mL ofdistilled water.

Caffeine (anhydrous; Sigma Reference Standard Product IC-1778) was usedas a standard in a caffeine-free beverage (product specific) as aninternal calibration standard for the presence of caffeine. Caffeine hasexcitation maxima at 254 nm and 330 nm and an emission maximum at 350nm.

Compound 1 and Compound 3 both require a 52OBP±15 nm band pass filter.Caffeine requires a 345LP nm long pass filter. Compound 11 requires a365B+15 band pass filter. Compound 12 requires a 46OBP+6.8 nm band passfilter. Compound 13 requires a 550BP+15 nm band pass filter.

Combination 2, for analyzing diet carbonated beverages is essentiallythe same as Combination 1 except that Compounds 1, 3, and 11 arereplaced by light-emissive compounds that indicate the relative presenceof aspartame. These include light-emissive compounds that react, orinteract, with carboxylic acid groups and amine groups. See Table 1 forexamples.

EXAMPLE 5

Infant Formulas

The methods of the invention can be used in the infant formula industryas for product authentication. In 1995, a counterfeit-labeled version ofinfant formula was illegally distributed to grocery chains in 16 states.Authenticating infant formula on shelves can help assure formulacustomers that a product is authentic and reliable, Using the methods,one can insure that the product at the source matches the product at thedestination. In addition, it can be possible to detect product tamperingby, fingerprint analysis.

Product formulations can be verified by methods similar to thosedescribed in Example 1. The fingerprint is the same when the product isproduced to the same high quality of standards. Referring to Table 7,light emission was monitored in an array of six samples (Gerber, Similacliquid, Similac powder, Carnation Follow-Up, Enfamil, and a powderedmilk standard) that were each tested four times with a light-emissivecompound. The light-emissive compound included Compound 1, Compound 2,Compound 3, and Compound 7.

The samples were prepared by diluting the infant formulas with distilledwater according to manufacturer instructions (e.g., 8.5 grams in 60 mLof distilled water). The resulting solutions were further diluted by afactor of 1000, and filtered using a Millipore 0.22 /μm sterile syringefilter. The filter samples were used directly in the analyses.

Stock solutions of the light-emissive compounds were prepared thatcontained Compound 1 at a concentration of 1.5 mM in a 1:2 DMSO/watermixture, Compound 2 at a concentration of 2 mM in DMSO, Compound 3 at aconcentration of 1 mM in DMSO, and Compound 7 at a concentration of 1 mMin distilled water.

Working solution concentrations were determined as described inExample 1. The working solution of Compound 1 was prepared by diluting75 μL of the stock solution in 20 mL of distilled water. The workingsolution of Compound 2 was prepared by diluting 120 μL of the stocksolution in 20 mL of distilled water. The working solution of Compound 3was prepared by diluting 100 μL of the stock solution in 20 mL ofdistilled water. The working solution of Compound 7 was prepared bydiluting 25 μL of the stock solution in 50 mL of distilled water.

Compounds 1, 2, 3, and 7 require a 53OBP±15 nm band pass filter toreduce the excitation wavelength intensity in the emission measurement.The analysis was conducted as described in Example 1. The diluted andfiltered samples were added directly to the dyes and the emissionmeasured.

The results of the experiment are presented in Table 7. There was onemeasurement made for each sample in combination with the light-emissivecompound. Each measurement was repeated four times to demonstrate thelevel of reproducibility. Variance and mean were calculated for eachgroup of 4 measurements. The 95% confidence levels were used for thisfingerprint analysis. The analysis is similar to that described inExamples 1 and 2.

For example, the Gerber sample had a mean light emission of −0.2049 andthe Similac sample had a mean light emission of −0.1941. The differencein light emission was 0.0108. The omega for the test was 0.044. If thedifference (0.0108) is less than omega for any two samples, then thesamples are substantially the same. Therefore, the products aresubstantially the same. The fingerprint data are presented in Table 8 tomake all possible comparisons and are established in the same manner asin Example 1. As a result of the fingerprinting analysis in Table 8, theGerber, Similac and Enfamil samples are the same. However, the Carnationand Standard are different. The Standard is Carnation Evaporated Milk.

TABLE 7 Similac Similac Car- Stan- Gerber Lq Pw nation Enfamil dardMeas. 1 −0.2022 −0.1912 −0.2109 −0.3088 −0.2143 −0.1022 Meas. 2 −0.2025−0.2061 −0.2102 −0.3196 −0.1511 −0.1154 Meas. 3 −0.1988 −0.1814 −0.2410−0.3037 −0.2493 −0.1094 Meas. 4 −0.2159 −0.1979 −0.1926 −0.3162 −0.2005−0.1155 Va- 5.708 0.0001094 0.0004035 5.163 0.0016566 3.968 riance E-05E-05 E-05 Mean −0.2049 −0.1942 −0.2137 −0.3121 −0.2038 −0.1106 MSE =0.0195 OMEGA = 0.044

TABLE 8 Ger- Similac Similac ber Lq Pw Carnation Enfamil Standard Gerber0 0 0 1 0 1 Similac Lq 0 0 0 1 0 1 Similac Pw 0 0 0 1 0 1 Carnation 1 11 0 1 0 Enfamil 0 0 0 1 0 1 Standard 1 1 1 0 1 0

EXAMPLE 6

Vanilla Extract

The Federation of Extracts Manufacturing Association needs to determineorigin of vanilla extract. The technology of the present invention wasused to identify geographical origin of vanilla samples. To date therewas not a practical method for identifying geographical origin ofvanillas. Geographical origin can be verified by methods similar tothose described in Example 1. The fingerprint of a Vanilla extract isthe same when the sample comes from the same geographical region.Referring to the table entitled FEMA Vanilla Extracts Pilot Program #1,Vanilla prized as high value from Bourbon had a fluorescent valueranging from 0.57-7.76. In contrast, vanilla from Java, a lessor valuedextract, had a fluorescent value ranging from 9.42-13.57. The testmethods were generally conducted in the following manor. The vanillaextracts were diluted 1/20 with water for optimum reaction with thelight emissive compounds. The optimum response of the vanilla sample isdetermined empirically, by using a concentration curve to maximizeinfrared region emission response. Stock solutions of the light-emissivecompounds were prepared by dissolving Indocyanine green at aconcentration of 1.5mM in 1:2 DMSO/water right before use and are onlygood for approximately 2 hours kept at room temperature. The workingsolution of this compound was prepared by diluting 120 μl of the stockin 20 ml of distilled water. Indocyanine green has an excitationwavelength of 775 nm. The sample placement and emission analysis wascarried out as described in Example 1. The result of the experiment arepresented in table 9. There were four measurements of the vanillasamples in each geographical region made for each combination with eachlight emissive compound. If two geographical regions differ by an amountgreater than Omega, then the vanilla from the regions can bedistinguished. The Omega for the Indocyanine green dye #1, is 0.35 andtherefore Bourbon can be distinguished from Java.

TABLE 9 FEMA VANILLA EXTRACT PILOT PROGRAM VANILLA DYE #1 Developed forFEMA Sample GFM Sample GFM Sample GFM Sample GFM Bourbon #1 0.57 Bourbon#21 2.48 Madagascar #18 1.94 Madagascar #19 3.90 Madagascar #24 4.54Madagascar #26 5.30 Comoros #27 4.09 Bourbon #40 Comoros #11 5.60Comoros #12 5.89 Tonga #30 5.27 Tonga #16 6.50 Indonesian #17 5.50Indonesian #20 8.34 Bali #29 7.96 Bali #36 9.43 Java #13 9.42 Java #3810.33 Java #14 10.66 Java #6 13.57 OMEGA 0.35 Note: #s Refer to FEMACode

EXAMPLE 7

In this example, light emissive compounds 1 & 4 were used to identify apatented compound. A product from China has the same light emissivefingerprint. The chances of this happening due to chance alone for 4replicates repeated 1 time is 1:100, repeated two times 1:10,000 andrepeated three times is 1:1,000,000. Therefore these compositions haveboth the same compound and the same concentration of the compound in theformulation. It also can be inferred that the compounds weremanufactured by the same process. In this example the product wasdiluted 1:1000 w/w and filtered with a 0.22 μm filter to removeparticles. Compound 1 and compound 4 were prepared as describedpreviously. The results of this experiment are presented in tables 10and 11, below. There were four measurements made of the customer'scompound (Std#39). The Chinese products #7 and #10 had the samemeasurements as the standard, sample #39. The patented compound has thesame emissive response with dye 1 as the two “suspect formulation” i.e.81.9 not different from 79.6 or 79.7 with an omega of 3.2. Measuringusing another dye, the same emissive response as the two “suspectformulations” i.e. 23.97 is not different from 24.86 and 24.40 with anomega of 1.58.

TABLE 10 STATISTICAL DATA: Dye 1 Std #39 Chinese #7 Chinese #10Measurement 1 81.3 80.3 77.1 Measurement 2 81.0 79.1 80.7 Measurement 382.5 79.2 80.2 Measurement 4 82.7 79.9 80.7 Variance 0.6797 0.30683.1052 Mean 81.9 79.6 79.7 MSE 0.1364 OMEGA 3.2

TABLE 11 STATISTICAL DATA: Dye 4 FF w/Std #39 FF w/ Chinese #7 FF w/Chinese #10 Measurement 1 24.21 24.84 24.36 Measurement 2 25.14 25.1223.98 Measurement 3 23.54 24.74 24.38 Measurement 4 22.99 24.73 24.86Variance 0.09 0.00 0.01 Mean 23.97 24.86 24.40 MSE 0.03 0.00 0.00 OMEGA1.58 0.00 0.00

Other embodiments are within the claims:

What is claimed is:
 1. A method for selecting a dye for determiningauthenticity of a product, comprising, combining a candidate dye with aplurality of candidate dilutions of a liquid sample of an authenticstandard of the product, said candidate dye being light emissive at aparticular wavelength when irradiated if it interacts with an analyte inthe liquid sample, selecting a test dilution at which said candidate dyeemits light at a selected intensity when said candidate dye is combinedwith said liquid sample at the test dilution, determining a range ofintensity of light emission for a plurality of mixtures of saidcandidate dye and said liquid sample at said test dilution, determiningan experimental intensity of light emission for a mixture of saidcandidate dye and a liquid sample of a nonauthentic product at said testdilution, and, comparing the experimental intensity to said range, saiddye being selected as useful for determining authenticity of saidproduct if said experimental intensity falls outside of said range. 2.The method of claim 1, wherein the candidate dye is a plurality ofcandidate dyes, each of said dyes emitting light over particularwavelengths, and wherein the analyte is a plurality of analytes, eachdye binding to a different of said plurality of analytes.
 3. The methodof claim 1, wherein the product is a liquid consumable product.
 4. Themethod of claim 2, wherein the product is a liquid consumable product.